Download THYMIC REQUIREMENT FOR CYCLICAL IDIOTYPIC AND

Published February 1, 1980
T H Y M I C R E Q U I R E M E N T F O R CYCLICAL IDIOTYPIC AND
R E C I P R O C A L ANTI-IDIOTYPIC I M M U N E RESPONSES T O A
T - I N D E P E N D E N T ANTIGEN*
By GARNETT KELSOE,~ DALE ISAAK,§ AND JAN CERNY
From the Departments of Tropical Public Health and Microbiology, Harvard School of Public Health,
Boston, Massachusetts 02115
* Supported in part by American Cancer Society grant IM-35 and by U. S. Public Health Service grant
RO1-CA-14922, and contract NO1-CB-64035 from the National Cancer Institute.
:~Recipient of a Rockefeller Foundation Fellowship. Present address: Institute for Genetics, University
of Cologne, Cologne, Federal Republic of Germany.
§ Supported by U. S. Public Health Service Fellowship 1 F32 CA 06122-01 from the National Cancer
Institute. Present address: Department of Microbiology and Immunology, Kirksville Collegeof Osteopathic
Medicine, Kirksville, Mo.
1Abbreviations used in this paper: AFC, antibody-forming cells; BBS, borate-buffered saline; FCS, fetal calf
serum; HA, hemagglutination; HBSS, Hanks' balanced salt solution; LM, phenotypically normal littermates of nude mice; M-315, the idiotype of the myeloma protein secreted by the MOPC-315 plasmacytoma;
M-603, the idiotype of the myeloma protein secreted by the McPC-603 plasmacytoma; PFC, plaqueforming cells; Pn, Streptococcuspneumoniae, strain R36a; PnC, cell wall polysaccharide purified from S.
pneumoniae; PnC-SRBC, sheep erythrocytes coated with PnC; TG-nude, thymus gland-reconstituted nude
mice; T-15, the dominant idiotype of the myeloma protein secreted by the TEPC-15 plasmacytoma; TNP,
trinitrophenyl; TNP-SRBC, TNP-substituted sheep erythrocytes; saline, 0.15 M NaCI; SRBC, sheep
erythrocytes.
J. Exp. MED.© The Rockefeller University Press • 0022-1007/80/02/0289/12 $1.00
289
Volume 151 February 1980 289-300
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T h e cyclical appearance of antibody-forming cells (AFC) 1 in the spleen and
peripheral blood following a single antigenic stimulus is a well-documented phenomenon (1). Generally, within the 2-wk period after an antigen stimulus, two peaks of
AFC are observed. T h e first peak occurs at ~ 4 - 6 d and the second at 10-13 d (1-8).
Both T-independent (2-4) and T - d e p e n d e n t antigens (5-8) are capable of inducing
very similar cyclical A F C responses in a wide variety of organisms.
Various explanations for the cyclic production of A F C have been offered including:
competition between free a n t i b o d y and A F C precursors for persistent antigen (2, 6);
sequential expression of various classes of antigen-specific immunoglobulin (5); the
release of antigen by macrophages (2); regulation of A F C formation that is complement dependent (3); synchronous recruitment of m e m o r y B cells that is regulated by
highly localized levels of specific a n t i b o d y (7); and regulator T cells (8).
Recently, results from our laboratory have indicated that the cyclical appearance
of splenic plaque-forming cells (PFC) m a y be the result of cell-to-cell interactions
based upon idiotypic recognition (4). We observed a cyclical production of splenic
PFC after a single injection of heat-killed strain R36a Streptococcus pneumoniae (Pn) in
B A L B / c mice. Concurrent with the anti-Pn response was a cyclical and reciprocal
expansion (or activation) of the clone(s) recognizing the d o m i n a n t idiotype of the
m y e l o m a protein secreted by the T E P C - 1 5 p l a s m a c y t o m a (T-15) of the Pn-stimulated
lymphocytes.
As both T and B cells are known to participate in anti-idiotypic responses, we
Published February 1, 1980
290
THYMIC REQUIREMENT FOR ANTI-IDIOTYPIC RESPONSES
Materials and Methods
Animals. BALB/c mice were purchased from Charles River Breeding Laboratories (Wilmington, Mass.). Congenitally athymic, nude (nu/nu) mice with the BALB/c genetic background and their phenotypically normal littermates (LM) (+/nu or + / + ) were obtained either
through a National Cancer Institute contract with Charles River Breeding Laboratories or were
from our own colony founded from mice provided by Dr. Norman Reed, Montana State
University, Bozeman, Mont. The latter are produced by heterozygous parents derived from the
ninth backcross on the BALB/c strain. Only mice from the same source were used in an
individual experiment (Charles River Breeding Laboratories, exp. 1 and 2; and mice from our
colony, exp. 3).
Thymic reconstitution of nude mice was accomplished by implanting a single neonatal
BALB/c thymus beneath the capsule of each of the recipient's kidneys (thymus gland-reconstituted nude [TG-nude] mice) as described by Dukor et al (9). All TG-nude mice were examined
for evidence of a successful thymic graft at the time of the PFC assay. TG-nude mice were
rested at least 4 wk after thymic implantation and used within 6 wk post-implantation. All
mice within a single experiment were age-matched to ~ + 3 wk. Mice of both sexes were used.
Antigen and Immunizations. Pn was grown in Todd-Hewitt broth and harvested during
exponential growth according to the method of Liu and Gotschlich (I0). The recovered bacteria
were washed four times in 0.15 M NaCI (saline) and lyophilized in sterile vials (Wheaton
Scientific Div., Wheaton Industries, Millville, N. J.; catalog No. 224822). A bacterial vaccine
was prepared by reconstituting the contents of a vial with sterile distilled water and heating the
contents to 60°C for 45 min. No viable bacteria could be demonstrated after this treatment.
The suspension was then adjusted to a concentration of 100 #g bacterial lyophylate/ml saline.
This Pn vaccine was stored at 4°C for the duration of an experiment.
The time-course of the idiotypic (T-15) and anti-idiotypic (anti-T-15) clonal responses after
stimulation by Pn was visualized by the sequential immunization of groups (three to four) of
mice. Daily, for 14 d, a group of mice was immunized by an intraperitoneal injection of 20 p.g
of Pn vaccine. On the 15th d, all immunized mice and a control group of comparable (normal,
nude, or TG-nude) unimmunized mice were killed and their spleens and sera assayed as
described below. Thus each group represented a unique stage of the anti-Pn response.
Preparation of the Cell Wall Polysaccharide of S. pneumoniae (PnC)-coated Sheep Erythrocytes. PnC
was extracted according to the procedure of Liu and Gotschlich (10) with the exception that
sodium deoxycholate was used at a concentration of 1.0% and that purification was halted after
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wished to functionally a n a l y z e the role o f the two cell p o p u l a t i o n s in terms o f the
cyclical i m m u n e response. Thus, we s t u d i e d b o t h the i d i o t y p i c (antigen reactive) a n d
a n t i - i d i o t y p i c i m m u n e responses in normal, a t h y m i c (nude), a n d t h y m u s - g r a f t e d n u d e
mice after a single antigenic challenge with Pn. W e could c o m p a r e , by utilizing
n o r m a l a n d n u d e mice, the effects o f direct a n t i b o d y feedback m e c h a n i s m s a n d those
regulatory mechanisms r e q u i r i n g T cells u p o n the cyclical course o f b o t h the i d i o t y p i c
a n d a n t i - i d i o t y p i c i m m u n e response.
This s t u d y d e m o n s t r a t e s t h a t b o t h the cyclical idiotypic (antigen specific) a n d antiidiotypic i m m u n e responses are t h y m u s d e p e n d e n t in B A L B / c mice. N u d e mice
(backcrossed onto the B A L B / c genetic b a c k g r o u n d ) respond with only a single p e a k
o f a n t i - P n P F C t h a t is equal to the initial B A L B / c response. In b o t h groups of mice,
the T-15 clone is d o m i n a n t a n d c i r c u l a t i n g a n t i - P n a n t i b o d y titers are similar. T h e
reciprocal a n t i - i d i o t y p i c response does not occur in n u d e mice b u t t h y m i c reconstitution of n u d e mice restores the cyclical idiotypic a n d a n t i - i d i o t y p i c responses. These
results s u p p o r t the concept that the cyclical i m m u n e response is a p r o d u c t o f
regulatory interactions between idiotypic a n d a n t i - i d i o t y p i c l y m p h o c y t e clones a n d
that T ceils are required for the cyclical responses after i m m u n i z a t i o n with a T i n d e p e n d e n t antigen.
Published February 1, 1980
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291
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ether extraction and dialysis against cold, distilled water. This dialyzate was lyophilized and
stored at -20°C.
Sheep erythrocytes (SRBC), (Colorado Serum Co., Denver, Colo.) were coated with the
recovered PnC by a procedure similar to that for bacterial lipopolysaccharide described by
M~iller (11). Briefly, PnC was dissolved in saline to a concentration of 0.1-0.2 mg/ml. This
solution was heated in a boiling water bath for ~30 min, briefly cooled, and then added to
packed, washed (three times in saline) SRBC at a ratio of 0.5 ml PnC solution:0.1 ml packed
SRBC. The cells were then gently mixed with a Pasteur pipette and allowed to incubate with
the PnC solution for 30 min at 37°C. The cells were washed twice in a large excess (20- to 50fold) of saline and finally in Hanks' balanced salts solution (HBSS), pH 7.1.
The success of PnC coating was determined by specific hemagglutination (HA) using
clarified ascitic fluid from BALB/c mice bearing, intraperitoneally, the TEPC-15 plasmacytoma. The ascitic fluid did not agglutinate normal SRBC and the hemagglutination titer
against PnC-coated SRBC (PnC-SRBC) was always ~ 1:100,000.
Source and lodination of Myeloma Proteins. The TEPC-15, MOPC-315, and McPC-603
BALB/c plasmacytoma lines were obtained from Litton Bionetics (Kensington, Md.) under a
National Cancer Institute contract. Each tumor was maintained by subcutaneous passage in
BALB/c mice. Ascites fluid containing the relevant myeloma protein was obtained by injecting
mice intraperitoneally with 0.5 ml of mineral oil (Diamond Drug Incorporated, Westhaven,
Conn.) at least 1 wk before an intraperitoneal inoculation of tumor cells. The resulting ascitic
fluid was recovered, pooled, and clarified by centrifugation. The proteins contained in each
ascitic fluid were twice precipitated by the addition of an equal volume of saturated (NH4)2SO4.
This precipitate was dissolved in a small amount of water and extensively dialyzed against
borate-buffered (0.1 M) saline (BBS), pH 8.0.
The dialyzate was then further purified by gel filtration on a Sephadex G-200 (Pharmacia
Fine Chemicals, Div. of Pharmacia, Inc., Piscataway, N. J.) column equilibrated with BBS.
The eluate was monitored for absorption at 280 nm and for the ability to agglutinate antigencoated SRBC. A protein peak corresponding to the void volume of the Sephadex G-200 column
also contained most of the HA activity. The recovered TEPC-15 (IgA, ~; anti-phosphorylcholine) and McPC-603 (IgA, ~; anti-phosphorylcholine) myeloma proteins agglutinated PnCSRBC but not trinitrophenyl (TNP)-substituted (TNP-SRBC) (12) or normal SRBC. The
MOPC-315 myeloma protein (IgA, ~.z; anti-nitrophenyl) agglutinated only TNP-SRBC. The
recovered purified myeloma proteins were concentrated to ~20 mg protein/ml BBS and stored
at -60°C.
Radio-iodination of purified myeloma proteins was accomplished via the N-hydroxysuccinimide ester of mono-iodinated (lzsI)p-hydroxyphenylpropionicacid (13), obtained from New
England Nuclear (Boston, Mass.). Iodination was carried out generally according to the method
of Bolton and Hunter (14). Briefly, a volume (--<500/xl) of benzene containing = 1.0 mCi of 125I
(-1,500 Ci/mmol) was transferred to a chilled (~5°C) vial and the benzene evaporated with
a stream of dry N2 gas. The vial was then cooled to 0°C and 5 mg of myeloma protein added
(10 mg/ml BBS). The reaction was allowed to continue for 1.5-2 h at 0°C with continuous
stirring. At this time, 0.5 ml of 0.2 M glycine (in BBS) was added, destroying any unreacted
iodination reagent.
The iodinated myeloma proteins were separated from any contaminating glycine (and/or
glycine conjugates) and 3-(4-hydroxyphenyl) propionic acid by gel filtration over Sephadex G50. Columns were equilibrated with 0.05 M phosphate buffer, pH 7.5, and = 10 mg of bovine
serum albumin was washed through before the addition of the iodination reaction products.
Two peaks of radioactivity were recovered, the first coinciding with the void volume of the
column. This first peak was dialyzed against continuously flowing phosphate-buffered saline
for 48 h at 4°C. The sp act of the recovered dialyzate was --~6-8 × 104 cpm//~g protein and
__.93% of this activity was precipitated by trichloroacetic acid. The ability of the radiolabeled
myeloma proteins to hemagglutinate the appropriate antigen-coupled SRBC was the equivalent
of unlabeled proteins.
Serum Antibody Assay. Mice were killed by decapitation and ~0.4 ml of blood was collected
from each mouse. The collected blood was allowed to clot overnight at 4°C. Serum was then
harvested from each tube, heat inactivated, and absorbed with SRBC. Finally, a 50-p.l serum
Published February 1, 1980
292
THYMIC REQUIREMENT FOR ANTI-IDIOTYPIC RESPONSES
T- 15 bound (percentage of control)
= (counts per minute T-15 immunized) - (counts per minute M-603 immunized)
× I00.
(counts per minute T-15 control) - (counts per minute M-603 control)
Specifically, 5/~g of radiolabeled myeloma protein (preliminary experiments indicated that
1 /~g of either TEPC-15 or MOPC-315 saturated the binding capacity of 106 BALB/c
splenocytes [4]) was added to duplicate tubes containing 106 splenocytes in ice-cold HBSS
containing 2% FCS. The tubes were incubated overnight at 4°C. The cells were then washed
four times with cold HBSS containing 10% FCS and the resultant cell pellets counted in a
Packard y-spectrometer (Packard Instrument Co., Inc., Downers Grove, Ill.). The cpm/tube
ranged from ~-5 × 103 to 12 × 103 cpm, representing ~60 ng of bound myeloma protein. The
variation between duplicates was routinely ~12%.
Only a single control (the idiotype of the myeloma protein secreted by the MOPC-315 or the
McPC-603 plasma cytoma) idiotype was used in a given experiment. Previous experiments
demonstrated that either protein serves equally well as a control (4).
Inhibition of PFC by Heterologous Anti-T-15 Serum. Rabbit anti-T-15 serum was raised by
injecting purified TEPC-15 myeloma protein into rabbits. The antiserum was rendered specific
by absorption with pooled murine IgM and the purified MOPC-315 myeloma protein insolubilized onto Affi-gel 10 Sepharose beads (Bio-Rad Laboratories, Richmond, Calif.) and by
incubation with normal C57BL/6 splenocytes (10V/ml antiserum). The addition of this antiserum at a 1:500 final dilution inhibited by --85% the number of anti-Pn PFC produced by a
representative pool of Pn-immunized BALB/c mice. The numbers of either C57BL/6 anti-Pn
PFC or BALB/c anti-SRBC-PFC were not inhibited at the same concentration.
Statistical Analyses. In certain cases Student's t statistic (two-tailed) was employed to determine if significant differences (P _<0.05) existed between serial PFC values.
Results
T h e specificity of the labeled idiotype probes was d e t e r m i n e d by i n h i b i t i o n studies
utilizing u n l a b e l e d homologous or heterologous protein competitors (Fig. I). A d d i t i o n
of a 100-fold excess of u n l a b e l e d homologous protein resulted in a >50% decrease in
b i n d i n g of the labeled idiotype. T h e same excess of heterologous protein resulted in
only "'18% inhibition.
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sample was tested for its capacity to agglutinate PnC-SRBC. The initial dilution of each serum
was 1:4 and all titrations were performed in duplicate, using microtiter plates (Cooke Engineering Co., Alexandria, Va.).
Preparation of Lymphocytes. Immediately after blood collection, the spleen was removed into
a dish containing ice-cold HBSS. A suspension of splenocytes was then made by pressing the
spleen through a stainless steel screen. This suspension was transferred to a conical-bottomed
tube. Cell clusters and noncellular debris were removed by allowing the spleen cell suspensions
to sediment briefly and then transferring the supernates to a second tube. These cell suspensions
were then washed three times in cold HBSS containing 2% fetal calf serum (FCS) (Microbiological Associates). The washed cells were resuspended in 5 ml of HBSS containing 2% FCS,
the numbers of viable lymphocytes/ml determined by trypan blue exclusion, and the volume
of each cell suspension adjusted to a final concentration of 10v viable lymphocytes/ml.
PFC Assay. Aliquots of these suspensions were used to determine the numbers of anti-Pn
PFC/106 splenocytes. A modified hemolytic plaque assay in agarose (15) was employed utilizing
PnC-SRBC (4). The background of anti-SRBC PFC (routinely ___5/106 splenocytes) was
determined and that number subtracted from the anti-PnC-SRBC PFC count. Each determination was performed in duplicate.
Idiotype-bindin~ Assay. Cell-associated anti-idiotypic activity was determined as the ability of
an aliquot of 10 viable splenocytes to bind specifically the T- 15 idiotype. This was accomplished
by incubating the cells with either the radiolabeled TEPC-15 or a control myeloma protein
(McPC-603 or MOPC-315) and determining the cell-bound activity. Specific T- 15 binding was
calculated as:
Published February 1, 1980
G A R N E T T KELSOE, DALE ISAAK, AND JAN CERNY
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F]c. 2. The kinetics of primary immune response of BALB/c mice to Pn: (a) specific anti-Pn PFC
in the spleen; (b) relative changes in binding of 12SI-TEPC-15 by splenocytes. Each point represents
the mean from a cohort of three to four mice, in three successive experiments (17, I , and A).
The kinetics of both PnC-specific PFC production and relative T-15 binding by
spleen cells from Pn-immunized BALB/c mice were determined in three successive
experiments (Fig. 2). The accumulation of specific PFC after a single injection of
vaccine followed a cyclical pattern over a 14-d period; an initial relatively broad peak
(=400 PFC/106 splenocytes) occurred on day 4-6 declining variably thereafter until
a second sharper peak (= 150 PFC/106 splenocytes) appeared on day 12-13 (Fig. 2 a).
The pattern of T-15 binding was also cyclical, with heights of binding activity
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Fx6. 1. Inhibition of binding of radiolabeled myeloma proteins on BALB/c spleen cells (10 s) in
the presence of increasing amounts of competing unlabeled homologous or heterologous myeloma
proteins (abscissa). The amount of cell-bound activity is expressed as the percent of that in the
absence of any competitor. ]2~I-TEPC-15 (1 #g) plus TEPC-15 (r-I) or plus M o P e - 3 1 5 (O); 12~IM o P e - 3 1 5 (1/~g) plus MOPC-315 (11) or plus TEPC-15 (O).
Published February 1, 1980
294
THYMIC REQUIREMENT FOR ANTI-IDIOTYPIC RESPONSES
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Fla. 4. The kinetics of Pn-specific PFC responses in the spleens of.. (a) BALB/c mice ( I ) and LM
([ZI); and (b) nude mice (0). Each point represents the mean from a cohort of three to four mice
with standard deviations (SD) indicated by the vertical bar.
occurring on d a y 2-4, d a y 10-11, a n d d a y 14 (Fig. 2 b). This reciprocal out-of-phase
relationship between the curves of Pn-specific P F C a n d T-15 b i n d i n g is typical o f the
B A L B / c a n t i - P n i m m u n e response (4).
In contrast, the kinetics o f splenic a n t i - P n p r o d u c t i o n in thymus-deficient, n u d e
mice i m m u n i z e d with Pn vaccine was not cyclical (Fig. 3 b). Instead, a first p e a k of
specific P F C reached on d a y 5 (445 + 170 P F C / 1 0 6 splenocytes) was followed b y a
decline a n d then a p l a t e a u d u r i n g which the P F C values r a n g e d between 25 _ 13
(day 10) a n d 40 ± 15 (day 13). No significant second p e a k o f a n t i - P n response
occurred. T h e control g r o u p of e u t h y m i c B A L B / c mice d i s p l a y e d a typical b i p h a s i c
response (550 ± 160 P F C , d a y 5, a n d 225 ± 24 P F C , d a y 12) (Fig. 3a). S i m i l a r results
were o b t a i n e d in a n o t h e r e x p e r i m e n t c o m p a r i n g n u d e mice with b o t h L M a n d
B A L B / c mice (Fig. 4). T h e a n t i - P n P F C responses of e u t h y m i c mice were essentially
the same, with a first p e a k on d a y 5 - 6 followed b y a decline to near b a c k g r o u n d by
d a y 10 a n d a second p e a k a g a i n on d a y 12 (Fig. 4 a). N u d e mice, however, r e s p o n d e d
b y only one r o u n d of P F C c u l m i n a t i n g on d a y 5 (140 ± 20 P F C / 1 0 6 splenocytes)
(Fig. 4 b).
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Fro. 3. The kinetics of primary anti-Pn PFC response in the spleen of: (a) BALB/c mice (11), and
(b) nude mice (0). Each point represents the mean from a cohort of three to four mice with
standard deviations (SD) indicated by the vertical bar.
Published February 1, 1980
GARNETT KELSOE, DALE ISAAK, AND JAN CERNY
295
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F=c. 5. Anti-Pn antibody determined by passive HA in the serum of mice immunized with Pn
vaccine (20 #g intraperitoneally, on day 0). Each point represents the mean titer in a cohort of three
to four mice bled and killed on the indicated day after immunization: BALB/c ( ' ) , nude (O), LM
(I--1),and TG-nude (A) mice.
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Fie. 6. The kinetics of Pn-specific PFC responses in the spleens of.. (a) LM (C]) and TG-nude mice
(A); (b) nude mice (O). Each point is the mean ± SD from a cohort of four mice.
It should be noted that the circulating antibody response to Pn was indistinguishable between B A L B / c , nude, LM, and T G - n u d e mice as measured by Pn-specific
passive HA. Typically, the serum anti-Pn antibody titers would peak at day 8 and
thereafter decline. By day 14, all mice showed significantly decreased levels (20-30%
of m a x i m u m values) o f Pn-specific serum antibody (Fig. 5).
In all mice, the T-15 component of the anti-Pn response was d o m i n a n t as measured
by PFC inhibition. Pooled cells of representative B A L B / c , nude, and L M mice were
equally inhibited (75-85%) by a specific rabbit anti-T-15 serum. In cases where more
than a single anti-Pn PFC peak occurred, both were p r e d o m i n a n t l y T-15 positive.
T h e experiments shown in Figs. 3 and 4 were repeated using euthymic LM, nude
mice, and TG-nude. Again, the splenic anti-Pn PFC activity of L M mice was biphasic
with an initial peak on day 5-6 (140 _ 50 PFC/106 splenocytes) followed by a second
peak on day 12 (80 +_ 20 PFC/106 splenocytes) (Fig. 6 a). A biphasic pattern was not
apparent in nude mice (Fig. 6b). In this experiment, nude mice reached an initial
anti-Pn peak on day 5 (180 + 30 PFC/106 splenocytes) that declined significantly by
day 9. Anti-Pn values for day 9-14 ranged from a high of-~70 (day 10) to a low of
2 3 0 (day 12). No significant peak o f anti-Pn activity occurred. However, when nude
mice were grafted with genetically compatible B A L B / c thymus (TG-nude mice) and
immunized with Pn, the distinctive biphasic kinetics o f PFC production returned,
with peaks on days 4 (280 + 40 PFC/106 splenocytes) and 12 (75 + 35 PFC/106
splenocytes) (Fig. 6a).
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n
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Published February 1, 1980
296
T H Y M I C R E Q U I R E M E N T FOR ANTI-IDIOTYPIC RESPONSES
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Fxo. 7. Relative changes in binding of ]25I-TEPC-15 by spleen cells during'the anti-Pn antibody
response. Values are based upon the ratio of specific T-15 binding (Materials and Methods) between
immunized and nonimmunized mice. (a) BALB/c mice ([]), LM (i-q) and TG-nude mice (A); (b)
nude mice, exp. 1 (O) and exp. 2 (O).
Discussion
Our experiments demonstrate that the repeated (biphasic) appearance of anti-Pn
antibody (IgM) PFC in mice immunized with a single injection of Pn vaccine depends
on the presence of the thymus. Although Pn vaccine induces in nude mice an initial
peak of T-15-bearing PFC and levels of circulating anti-Pn antibody indistinguishable
from that occurring in normal mice, a biphasic PFC response is absent. Thymic
reconstitution restores the cyclical immune response. Thus, at least a portion of the
anti-Pn response seems to be thymus-dependent. Although our experiments cannot
rule out an effect of antibody that is mediated by T cells, it seems clear that serum
antibody does not affect the PFC precursors either directly or via competitive
interactions with antigen in such a way as to produce a biphasic anti-Pn response.
The immunization of BALB/c mice with Pn vaccine is accompanied by expansion
or activation of cells binding the dominant idiotype, T-15, of the Pn-reactive B cells.
Appropriate control experiments were performed to demonstrate that the increased
binding of 12~I-TEPC-15 is indeed specific for the idiotopic determinant and thus
reflects the amount of cell-associated T-15 receptors (i.e., anti-idiotypic cells). The
increased T-15-binding activity seen in BALB/c mice was completely absent in Pnimmunized nude mice. The latter could not be a result of a lack of idiotypic stimulus
because the initial response of T-1f-positive, anti-Pn PFC in nude mice is equivalent
to that in either BALB/c or LM mice. Furthermore, implantation of a thymus in
nude mice restores their ability to express the anti-idiotypic cellular activity.
Thus it would seem that although a sufficient idiotypic stimulus is present in nude
mice, a component necessary for the anti-idiotypic response is not. Ostensibly, this
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The changes in T-15 binding by splenic lymphocytes also differed between normal
and athymic mice. The data shown in Fig. 7 bear on the Pn response in Figs. 4 and
6. BALB/c, LM, and TG-nude mice showed patterns of T-15 binding that were
virtually identical (Fig. 7 a) and typical of the BALB/c anti-Pn immune response (4).
Peaks of T-15 binding occurred at day 3-4 and day 10-11. These expansions are the
reciprocal of the corresponding PFC kinetics. Conversely, T-15 binding in nude mice
either declined and remained depressed (Fig. 7 b, exp. 1) or did not seem significantly
affected by the ongoing anti-Pn response (exp. 2). The changes that did occur were
not in phase with the T-15 binding oscillations typical of t h e thymus-associated
BALB/c anti-Pn immune response.
Published February 1, 1980
GARNETT KELSOE, DALE ISAAK, AND JAN CERNY
297
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component is anti-idiotypic T cells. That both cyclical PFC production and the antiidiotypic response are missing in thymusless mice strongly implies that the cyclical
nature of the anti-Pn response is causally linked with the reciprocal anti-idiotypic
response. This is entirely consistent with the finding of Romball and Weigle (8) that
T cell and not B cell mitogens are capable of abrogating the cyclical immune response.
Cycling has been noted in various species with antigens ranging from haptens (16)
to bacterial antigen (2) and alloantigens (17), and in both T-independent and Tdependent antibody responses (2-8), as well as cell-mediated responses (18, 19). The
cycling is independent of the immunization route and of the serum antibody levels
(1-8). What is most remarkable is that the peaks of AFC appear almost exactly every
6-10 d regardless of the antigen or animal species. This constancy leads us to believe
that the cyclical immune response is the expression of a fundamental cybernetic
system for immunological regulation. Viewed in this light, our data appear to be a
significant demonstration of the idiotype-specific, regulatory equilibrium originally
postulated by Jerne (20-22).
Numerous studies have demonstrated that the capacity for idiotype-specific immune
regulation exists (23-29) most often by demonstrating idiotype-specific help, suppression, or activation. Our data are similar in that we demonstrate that specific activation
of the anti-T-15 clone(s) occurs during the BALB/c anti-Pn immune response. More
importantly, our data indicate that the interaction between idiotypic and antiidiotypic clones is thymus-dependent, mutually reciprocal, and continuing. There is
no reason to expect that this cycling ends after 14 d. Indeed, cyclical AFC production
has been shown to occur for at least 50 d (30) and regularly periodic fluctuation in
antibody production has been measured over a period of years (31). The longevity of
these cyclical phenomena seemingly preclude a role for exogenous antigen. Instead, it
is likely that the basis for continuing immune periodicity is entirely internal.
Recently, Eichmann et al. (32) have demonstrated that under the appropriate
conditions, anti-idiotypic T-helper cells can induce B-memory cells to become AFC,
even in the absence of antigen. It is conceivable that periodic waves of anti-idiotypic
helper cell activity could give rise to the long-lived cyclical immune response. The
cyclical immune response then, may be visualized as a dynamic regulatory equilibrium
that continuously balances idiotypic and anti-idiotypic cell populations via positive
and negative regulatory interactions.
The effectors of this regulation appear to be T cells. Thus, the cyclical immune
response is thymus-dependent. However, since Pn is a T-independent antigen, it is
likely that both T and B cells may induce those regulatory events leading to the
cyclical immune response. That primed B cells do have the capacity to induce T-cellmediated suppression has been demonstrated in other systems (33).
Finally, as it is known that the cyclical immune response may be abrogated by the
administration of passive specific antibody (30), the concept of anti-idiotypic regulation may complement previously proposed mechanisms of immune regulation, in
particular the antibody feedback effect. Even in those situations where the steady
level of circulating antibody ostensibly bears no relationship to the cycling appearance
of idiotypic or anti-idiotypic cells (4, 8, and this paper), significant cell-antibody
interactions might still occur in situ. Indeed, preliminary results indicate that T-15
binding increases after mild trypsinization and subsequent in vitro incubation of
splenocytes from Pn-immunized BALB/c mice (G. Kelsoe and J. Cerny. Unpublished
results.).
Published February 1, 1980
298
THYMIC REQUIREMENT FOR ANTI-IDIOTYPIC RESPONSES
Receivedfor publication 23July 1979 and in revisedform 16 October 1979.
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Summary
The role of the thymus in the cyclical appearance of the dominant idiotype of the
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